Vibrio cholerae is the causative agent of the devastating diarrheal disease cholera. This organism is also a natural inhabitant of aquatic environments worldwide. V. cholerae is thought to exist in these environments primarily in microbial communities called biofilms. Biofilm bacteria are more tolerant to environmental stresses such as UV radiation, desiccation, low pH, antimicrobials, and the immune defenses of the human host. This tolerance protects V. cholerae not only from the harsh conditions in the environment, but also from the acidic conditions of the host stomach. There is also evidence of V. cholerae biofilms in the host intestinal environment, which suggests that V. cholerae might use biofilm formation as a long-term protective strategy in the human host. Development of biofilms is controlled by signaling networks that respond to chemical signals in the environment. Our long-term goal is to identify and understand the signaling systems that regulate biofilm formation in V. cholerae. In this proposal, we focus our attention on a specific signaling system that responds to the polyamines norspermidine and spermidine. These polyamines are thought to act as environmental signals that are detected and processed by a signaling system composed of the proteins NspS and MbaA. NspS is predicted to be the sensor protein or the system that detects these polyamines. MbaA is predicted to be a phosphodiesterase that breaks down cyclic-diguanylate-monophosphate (c-di-GMP), one of the central modulators of biofilm formation in V. cholerae. We hypothesize that norspermidine and spermidine bind directly to NspS;these binding events affect the interaction of NspS and MbaA, which then results in changes in its c-di-GMP phosphodiesterase activity. This change is manifested as increases or decreases in c-di-GMP levels in the cell that lead to corresponding increases or decreases in biofilm formation. We base this hypothesis on the following observations: 1) NspS is homologous to the spermidine binding protein PotD of Esherichia coli;2) In the absence of NspS and MbaA, spermidine and norspermidine cannot affect biofilm formation when supplied extracellularly;3) MbaA contains an EAL domain, found in c-di- GMP phophodiesterases, in which all the residues that contribute to enzymatic activity are conserved. The goal of the proposed project is to provide definitive evidence for this signaling system. The specific aims of this project are to i) Establish that norspermidine and spermidine are ligands for NspS by doing binding experiments using isothermal titration calorimetry;ii) Provide evidence for NspS-MbaA interaction using coimmunoprecipitation experiments;and iii) Confirm that MbaA is a phosphodiesterase by enzyme assays and correlate this activity to its role in biofilm formation using biofilm assays. PUBLIC HEALTH RELEVANCE: Vibrio cholerae is the causative agent of the devastating diarrhoeal disease cholera. According to the World Health Organization, cholera is estimated to result in approximately 100,000 deaths worldwide every year. V. cholerae can exist in the human host in microbial communities called biofilms, which can make this organism highly tolerant to host defense mechanisms and potentially aid in disease progression (Hang et al., 2003, Xu et al., 2003). The insight gained from the proposed project, which involves studying V. cholerae biofilm formation, is expected to provide a better understanding of cholera and yield novel strategies to combat this deadly disease.